Lecture 3 Mutagens and Mutagenesis
1. Mutagens
A. Physical and Chemical mutagens
B. Transposons and retrotransposons
C. T-DNA
2.
A.
B.
C.
Mutagenesis
Screen
Selection
Lethal mutations
Read: 606-612
Figs:
14.10-13
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Transposon (transposable element) as a mutagen
Transposon: DNA segment that can move
from one position to another
(1) Retrotransposons
Copia
Drosophila (LTR-type)
Ty1
Yeast (LTR-type)
LINEs
Human (non-LTR-type)
SINEs (Alu)
Human (non-LTR-type)
(2) Transposons
Ac/Ds
Maize
P-element
Drosophila
2
Retrovirus
Figure 5-73. The life cycle of a retrovirus. The retrovirus genome consists of an RNA
molecule of about 8500 nucleotides; two such molecules are packaged into each viral
particle. The enzyme reverse transcriptase first makes a DNA copy of the viral RNA
molecule and then a second DNA strand, generating a double-stranded DNA copy of the
RNA genome. The integration of this DNA double helix into the host chromosome is then
catalyzed by a virus-encoded integrase enzyme. This integration is required for the
synthesis of new viral RNA molecules by the host cell RNA polymerase, the enzyme that
transcribes DNA into RNA. (From Molecular Biology of the Cell by Alberts et al.)
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4
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Figure 5-76. Transpositional sitespecific recombination by a nonretroviral
retrotransposon. Transposition by the L1
element (red) begins when an
endonuclease attached to the L1 reverse
transcriptase and the L1 RNA (blue)
makes a nick in the target DNA at the
point at which insertion will occur. This
cleavage releases a 3′-OH DNA end in
the target DNA, which is then used as a
primer for the reverse transcription step
shown. This generates a single-stranded
DNA copy of the element that is directly
linked to the target DNA. In subsequent
reactions, not yet understood in detail,
further processing of the single-stranded
DNA copy results in the generation of a
new double-stranded DNA copy of the L1
element that is inserted at the site where
the initial nick was made.
(Based on Molecular Biology of the Cell by
Alberts et al.)
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Fig. 13.24a
P-element in Drosophila
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Fig. 13.24b
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12
Transposons can insert into different regions of a gene
Fig. 13.25
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Ac/Ds are Maize transposons
Ac/Ds can function in other plants
Ac: activator, autonomous, 4.6 kb long, enocdes a
3.5 kb transcript of transposase
Transposase
Ds: dissociation, non autonomous
-both Ac and Ds have 11 bp inverted repeats at the ends,
which function in the transposase recognition
-an 8 bp direct repeat generated from the host genome-footprint
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Fig. 13.22
Ac/Ds in Corn Kernels
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Fig. 13.20
Barbara McClintock
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T-DNA for Plant transposon-tagging
Transposon mutagenesis facilitates gene cloning
1. T-DNA (transfer DNA) from Agrobacterium tumefaciens
Agrobacterium causes crown-gall diseases in plants. This tumor-inducing (Ti)
ability is linked to the Ti-plasmid.
During the infection, a segment of the Ti-plasmid, the T-DNA, is transferred into
the plant cell and integrated into the plant genome.
LB
RB
NPT-II
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Principles of gene transfer from Agrobacteria into plant cells
nucleus
T-DNA
Tiplasmid
host
chromosome
vir
Agrobacteria
Plant Cell
vir: vir region (vir = virulence)
Ti: tumor-inducing plasmid
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2. Mutagenesis
Screen:
Visual: flower morphology, pigment color,
Biochemical pathway mutant
auxotrophy: his3- in yeast; arg- in Neurospora; trp1- in plant
Reporter gene expression (luc bioluminescent protein from firefly)
Selection:
trp pathway: 5-methylanthranilate ---> 5’methyltrp (toxic)
ADH (alcohol dehydrogenase): Allyl alcohol---> acrolein aldehyde (toxic)
Lethal mutations:
(such as house keeping genes)
-maintain as heterozygote
-weak hypomorph
-conditional such as temperature-sensitive mutants in T4 phase (p220-221)
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WT Arabidopsis flower
ap2-2 floral mutant
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sex-determination mutant
(tsl2 =tassel seed 2)
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Fig. 1.11
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22
Fig. 7.14
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Fig. 5.21
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Fig. 7.20 a
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Tryptophan biosynthetic pathway
Chorismate
trp4 (AS)
Anthranilate (blue fluorescent under UV)
trp1(PAT)
Phosphoribosyl (PR)
anthranilate (blue fluorescent under UV)
PAI
CDRP
Indole-3-glycerol phosphate
Indole
L-tryptophan (amino acid)
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RD29A promoter
firefly luciferase (Luc)
Before stress
NPT-II
After stress
Control
PC-Luc
RD29A-Luc
Mutagenizes RD29A-Luc transgenic plants
to look for mutants that stop fluorescence under cold stress
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Fig. C.8
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